小脑颗粒细胞轴突起始段的FGF12-Nav1.2/Nav1.6相互作用改变导致PKD的机制研究

Paroxysmal kinesigenic dyskinesia (PKD) is a kind of synaptic disease related to ion channels. The known PKD genes can be verified in about 50% patients. Previous studies have identified FGF12 gene as a novel causative gene of PKD. The result of in vitro experiments by using co-immunoprecipitation and whole-cell patch clamp technique found that FGF12 mutant-Nav1.2/Nav1.6 is uncoupling, which leads to sodium channel currents increased. To further clarify the pathogenesis of truncated FGF12, we will firstly verify the interaction between FGF12 and Nav1.2/Nav 1.6, and then construct a FGF12 silencing model in cerebellar granule cells by shRNA-mediated gene silencing and transgenic technology. The co-localization at the axon initial segment and expression of FGF12, Nav1.2 and Nav1.6 in cerebellar granule cells between different groups were determined at the levels of mRNAs, proteins and cells, respectively. Finally, the whole-cell patch clamp was used to record the current density and kinetic changes of sodium channel. The purpose of this project was to study the effects of interaction changes between FGF12 and Nav1.2/1.6 on the distribution of Nav1.2 and Nav1.6 at the AIS and the neuronal excitability. It is very significant to reveal the pathogenesis of PKD and to find effective preventive and therapeutic methods.

发作性运动诱发性运动障碍（PKD）是一种可能与离子通道密切相关的“突触病”。目前已知致病基因仅在约50%的PKD患者得到验证。课题组前期发现并已成功克隆出新的PKD致病基因FGF12，基于免疫共沉淀和细胞膜片钳实验证明FGF12基因突变后与Nav1.2/Nav1.6失去正常的相互作用，导致钠离子通道电流增加。为进一步明确FGF12截短蛋白的致病机制，本课题拟在多重验证其蛋白相互作用的基础上，采用shRNA基因沉默和转基因技术，在原代小脑颗粒细胞中分别从mRNA、蛋白质和细胞水平检测不同组别间FGF12、Nav1.2和Nav1.6在轴突起始段的共定位、表达变化以及钠离子通道动力学改变。研究FGF12-Nav1.2/Nav1.6的相互作用改变对神经元AIS部位Nav1.2和Nav1.6的分布及神经元兴奋性的影响，对揭示PKD新的发病机理、寻找有效的预防和治疗手段具有极其重要的临床意义。

发作性运动诱发性运动障碍（PKD）的发病机制目前尚不明确，目前已知的致病基因仅在约50%的PKD患者中得到验证。根据基因功能考虑为单纯的突触病（如PRRT2）、离子通道病（如SCN8A、KCNA1）或基底节病变均尚不足以解释PKD发生和终止的整个病理过程。本项目首先基于PKD患者的结构和功能影像学分析，从宏观网络层面揭示了PKD的发生可能与小脑-丘脑-皮层环路功能异常密切相关，其中小脑是关键的起始部位。在前期发现并克隆了新的PKD致病基因FGF12的基础上，采用CRISPR/Cas9技术成功构建了Fgf12 c.472delC点突变小鼠模型。为了进一步明确FGF12截短蛋白的致病机制，在突变小鼠中分别从整体、细胞和分子三个不同层面研究Fgf12-/-、Fgf12+/-和WT三组小鼠间行为学、神经元兴奋性和突变蛋白表达分布的差异。结果发现：1）Fgf12-/-小鼠存在运动障碍样症状，且PTZ致痫阈值明显降低；2）Fgf12基因在小脑浦肯野细胞中高表达，突变后的Fgf12基因在大脑皮层和小脑中的表达量均明显增高；3）脑片钳实验显示Fgf12基因突变后小脑浦肯野细胞的放电频率明显增高，以Fgf12-/-小鼠更为显著；4）细胞膜片钳实验进一步证实神经元兴奋性增高可能与Fgf12增强了Nav1.2和Nav1.6的通道功能密切相关。FGF12突变介导的小脑皮层神经元兴奋性增高及小脑-丘脑-皮层神经环路的结构和功能障碍可能是PKD一个全新的发病机制。这一系列研究成果为PKD深入的致病机制研究提供了新的理论和实验范式，对于开发新的药物治疗靶点和寻找有效的预防和治疗手段亦具有极其重要的临床意义。

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